Project Summary Tendinopathy is a common chronic tendon disorder that affects 30-50% of individuals over 60 years old. It is characterized by pain, swelling, loss of function, and impaired performance. There is currently no cure for tendinopathy. Spontaneous repair or treatment typically leads to scar formation, resulting in a weakened tissue with reduced function and mechanical properties that may ultimately rupture with further use. Our previous and preliminary studies show that reprogramming tendon stem progenitor cells (TSPCs) derived from aged donors to increase their levels of CITED2 (TSPCCITED2) reverses the age-induced changes in the stem cells and enhances their reparative efficacy in rat models of tendon rupture. Moreover, intra-tendinous injection of aged TSPCCITED2 into the diseased tendon mitigated disease progression and relieved pain in a rat model. However, acquisition of TSPCs from patients is not feasible due to concerns regarding donor site morbidity. Adipose- derived stem cells (ADSCs) have multiple-lineage differentiation potential, including that of tendon, and is a patient-friendly source of mesenchymal stem cells (MSCs). Of interest, our pilot study suggests that CITED2 reprogramming may exert a similar effect on ADSCs as in TSPCs. In addition, aged ADSCs transferred with CITED2 (ADSCCITED2) and injected into the supraspinatus tendon of healthy recipients can survive for at least 14 days. Together, these findings led us to propose and test the hypothesis that aged ADSCs transferred with CITED2 (ADSCCITED2) exert an enhanced therapeutic effect on mitigating tendinopathy pathology and relieving tendinopathy-related pain. ADSCs and TSPCs isolated from aged and young Fischer rats ubiquitously expressing green fluorescence protein (GFP) will be subjected to gene transfer of CITED2 (ADSCCITED2 or TSPCCITED2) or vector control (ADSCV or TSPCV). Adult non-GFP-expressing Fischer rats subjected to overuse treadmill running at a stage of moderate tendinopathy will be subjected to one intra-tendon injection of aged ADSCCITED2 at two dosages, or injected with aged ADSCV, young ADSCV, aged TSPCsCITED2, aged TSPCV, and young TSPCV all as controls. A sham/placebo group (rats placed on treadmill without running and injected with a vehicle with no cells) will serve as an additional control. At 4 weeks after injection, supraspinatus tendons will be dissected and subjected to assays for: 1) histologic evaluation, 2) immunohistochemistry for tendon phenotypic proteins, and 3) mechanical property testing. We will also evaluate: 4) pain behaviors, and 5) pain- related pathogenic changes in the tendon and in the dorsal root ganglion (DRG). Upon successful completion of Phase I studies, using larger animal models (i.e., rabbit model of collagenase-induced tendinopathy) in Phase II studies, we will: 1) finalize the quality control protocol 2) optimize dose and treatment frequency. Animal models in Phase II studies will include tendinopathy of other commonly affected tendons, including the Achilles and patellar tendons, and these tendons will be the primary focus of future clinical trials. 1